Anti-Leukemic Effect of Maintenance Chemotherapy Is Mediated by Induction of Differentiation.

The clinical benefit of maintenance therapy (prolonged post-remission low-dose therapy) has been limited to two diseases, acute lymphocytic leukemia (ALL) and acute promyelocytic leukemia (APL), despite extensive study in many malignancies. Moreover, the mechanisms responsible for the anti-leukemic effects of maintenance therapy in both ALL and APL are unclear. Maintenance therapy in ALL consists of the low-dose cytotoxic agents methotrexate (MTX) and 6-mercaptopurine (6MP), while maintenance therapy in APL has primarily been all-trans-retinoic acid (ATRA). Interestingly, the addition of low-dose MTX and 6MP to maintenance ATRA has recently been shown to improve disease-free survival in APL. These clinical results, combined with our recent data and that from others showing that low-dose cytotoxic agents are potent differentiating agents, suggest that these agents may induce the differentiation of leukemic progenitors responsible for disease relapse. Therefore, we examined the differentiating effects of ATRA and low-dose MTX and 6MP on human APL and ALL cells. The APL cell line NB4 was treated with ATRA (1 μM), MTX (0.01 μM) and 6MP (1 μM) for 48 hours and then analyzed for apoptosis, clonogenic growth and surface expression of the myeloid differentiation antigens CD11b and CD15 by flow cytometry. As expected, ATRA did not induce immediate apoptosis, but inhibited clonogenic growth associated with markedly increased expression of both myeloid antigens, consistent with induction of terminal differentiation. Both MTX and 6MP had similar effects on the NB4 cells, and, thus, similarly appeared to induce terminal differentiation rather than cytoxicity. We then examined the effects of these agents on two ALL cell lines: REH, which is derived from pediatric ALL and contains the t(12;21) chromosomal translocation producing the TEL-AML1 fusion protein, and RS4;11, which is derived from adult ALL and contains the t(4;11) chromosomal translocation producing the MLL-AF4 fusion gene. ATRA, MTX and 6MP induced upregulation of CD19 and CD38 surface antigen expression. Similar to their effects on the NB4 APL line, the agents also inhibited clonogenic growth of the ALL cells without induction of immediate apoptosis. ATRA, MTX, and 6MP produce similar effects on APL and ALL cells in vitro. These results suggest that low-dose MTX and 6MP used as maintenance therapy improve clinical outcomes in ALL and APL by inducing the terminal differentiation of leukemic progenitors responsible for disease relapse. As the addition of low-dose MTX and 6MP has improved the results of maintenance ATRA in APL, the addition of pharmacologic differentiating agents such as ATRA to MTX and 6MP may improve ALL therapy, particularly for adults.